Triple glucagon/glp-1/gip receptor agonist

ABSTRACT

The present invention relates to a triple agonist having activities to all of glucagon, GLP-1, and GIP receptors and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a bypass continuation of Application No. PCT/KR2016/015554 filedDec. 30, 2016, which claims priority from Korean Patent Application Nos.KR 10-2016-0163737 filed Dec. 2, 2016 and KR 10-2015-0191082 filed Dec.31, 2015, of which entire disclosures are hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to a triple agonist having activities toall of glucagon, GLP-1, and GIP receptors, and uses thereof.

BACKGROUND ART

Obesity and diabetes including type 2 diabetes are representativemetabolic diseases that occur in modern society. These diseases areregarded as health-threatening factors in the world and the accompanyingeconomic costs due to the incidence of these diseases are rapidlyincreasing at present.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropicpolypeptide (GIP) are representative gastrointestinal hormones andneuronal hormones, and are materials involved in the control of bloodglucose levels according to food intake. Glucagon is a peptide hormonesecreted by the pancreas and is involved in controlling the bloodglucose levels along with the two materials described above.

GLP-1 is a hormone secreted by the small intestine stimulated by foodintake. GLP-1 promotes insulin secretion in the pancreas in a bloodglucose-dependent manner and inhibits the secretion of glucagon, thushelping the action of lowering blood glucose levels. Additionally, GLP-1has the roles of slowing digestive action in the gastrointestinal tractby acting as a satiety factor, and reducing the amount of food intake bydelaying the time for emptying digested food in the gastrointestinaltract. Furthermore, the administration of GLP-1 to rats was reported tohave effects of inhibiting food intake and reducing body weight, andthese effects were confirmed to occur equally both in normal and obesestates, thus showing the potential of GLP-1 as an agent for treatingobesity.

GIP, one of the gastrointestinal hormones secreted by the stimulation offood intake, as is the case of GLP-1, is a hormone consisting of 42amino acids secreted by the intestinal K-cells. GIP was reported toperform the functions of promoting the secretion of insulin in thepancreas in a blood glucose-dependent manner and helping to lower theblood glucose levels, thereby exhibiting the effects of increasing theactivation of GLP-1, anti-inflammation, etc.

Glucagon is produced in the pancreas when the blood glucose levels falldue to reasons such as medications, diseases, deficiency in hormones orenzymes, etc. Glucagon sends a signal for glycogen breakdown in theliver to induce the release of glucose and increases blood glucoselevels to a normal level. In addition to the effect of increasing theblood glucose levels, glucagon suppresses appetite in animals and humansand activates hormone-sensitive lipase of adipocytes to promotelipolysis and energy expenditure, thereby showing an anti-obesityeffect.

As such, active studies are being conducted to develop GLP-1 as atherapeutic agent for treating diabetes and obesity, based on theeffects of GLP-1 controlling blood glucose levels and reducing bodyweight. Currently, exendin-4, prepared from lizard venom and having anamino acid homology of about 50% with GLP-1, is under development as atherapeutic agent for treating the same kinds of diseases. However, thetherapeutic agents containing GLP-1 and exendin-4 were reported to showside-effects such as vomiting and nausea (Syed Y Y., Drugs, 2015 July;75 (10): 1141-52).

Additionally, for the maximization of body weight reduction and as analternative to the above-described GLP-1-based therapeutic material,studies have been focused on dual agonists having activities to bothGLP-1 receptors and glucagon receptors, and they were shown to be moreeffective in body weight reduction due to the activation of glucagonreceptors, compared to when the existing GLP-1 was treated alone(Jonathan W et al., Nat Chem Bio., 2009 October (5); 749-757).

Additionally, in the study related to triple agonists, which haveactivities to all of GLP-1, GIP, and glucagon receptors simultaneously,efforts have been made recently to increase the half-life of the tripleagonists by substituting an amino acid sequence to increase theresistance to dipeptidyl peptidase-IV (DPP-IV), which decomposesgastrointestinal hormones to get rid of their activities, followed byadding an acyl group to a particular region thereof (Finan B et al., NatMed., 2015 January; 21 (1): 27-36). However, their effects of activatingthree different kinds of receptors were not significant and no tripleagonist showed various active ratios thereto.

Accordingly, there is a need for the development of a novel materialwhich can highly activate GLP-1, GIP, and glucagon receptors and has theeffects of controlling blood glucose levels and reducing body weightwithout causing any side-effects such as vomiting and nausea.

Additionally, there is also a need for the development of a novelmaterial which has various active ratios to GLP-1, GIP, and glucagonreceptors. For example, there is an increasing need for the developmentof a material which has an effect of reducing body weight but has asignificantly higher effect of controlling blood glucose levels due tohigh GLP-1 and GIP activities but with relatively low glucagon activityfor a hypoglycemic effect; or a material which has high activities forall of GLP-1, GIP, and glucagon, thus having a significantly high effectof reducing body weight.

DISCLOSURE Technical Problem

An object of the present invention is to provide an isolated peptidehaving activities to a glucagon receptor, a glucagon-like peptide-1(GLP-1) receptor, and a glucose-dependent insulinotropic polypeptide(GIP) receptor.

Another object of the present invention is to provide a polynucleotideencoding the isolated peptide, a recombinant expression vector includingthe polynucleotide, and a transformant including the polynucleotide orrecombinant expression vector.

Still another object of the present invention is to provide a method forpreparing the isolated peptide.

Still another object of the present invention is to provide acomposition containing the isolated peptide.

Still another object of the present invention is to provide a method fortreating a target disease, which includes administering the isolatedpeptide or a composition containing the isolated peptide to a subject inneed thereof.

Still another object of the present invention is to provide a use of theisolated peptide or the composition for use in the preparation of amedicament.

Technical Solution

To achieve the above objects, in an aspect, the present inventionprovides an isolated peptide having activities to a glucagon receptor, aglucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependentinsulinotropic polypeptide (GIP) receptor.

In a specific embodiment, the peptide is an analog of native glucagonwith a variation selected from the group consisting of substitution,addition, deletion, modification, and a combination thereof, on at leastone amino acid of the native glucagon sequence.

In another specific embodiment, the amino acid sequence with addition isderived from a native GLP-1 amino acid sequence, a native GIP amino acidsequence, or a native exendin-4 amino acid sequence.

In still another specific embodiment, the peptide is an isolated peptideincluding an amino acid sequence represented by General Formula 1 below:

Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General Formula 1, SEQ ID NO: 103)

wherein, in General Formula 1,

Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y);

Xaa2 is glycine (Gly, G), α-methyl-glutamic acid, or Aib(aminoisobutyric acid);

Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q);

Xaa7 is threonine (Thr, T) or isoleucine (Ile, I);

Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine(Cys, C), or valine (Val, V);

Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I);

Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), orcysteine (Cys, C);

Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);

Xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic acid (Glu,E), or leucine (Leu, L);

Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);

Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I),glutamic acid (Glu, E), cysteine (Cys, C), or lysine (Lys, K);

Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), orhistidine (His, H);

Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C), orvaline (Val, V);

Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);

Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L),cysteine (Cys, C), or aspartic acid (Asp, D);

Xaa23 is isoleucine (Ile, I) or valine (Val, V);

Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C),asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E);

Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), ormethionine (Met, M);

Xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala, A),asparagine (Asn, N), or aspartic acid (Asp, D);

Xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine (Gln, Q),threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H);

Xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys, K), orhistidine (His, H), or is absent; and

R1 is cysteine (Cys, C), GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent;

wherein,

m is -Cys-, -Pro-, or -Gly-Pro-,

n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent.

In still another specific embodiment,

in General Formula 1,

Xaa14 is leucine or methionine; and

Xaa15 is cysteine, aspartic acid, or leucine.

In still another specific embodiment, in General Formula 1,

Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

Xaa7 is threonine;

Xaa10 is tyrosine, cysteine, or valine;

Xaa12 is lysine or isoleucine;

Xaa13 is tyrosine, alanine, glutamine, or cysteine;

Xaa14 is leucine, cysteine, or methionine;

Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;

Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, orlysine;

Xaa18 is alanine, glutamine, arginine, or histidine;

Xaa19 is alanine, glutamine, valine, or cysteine;

Xaa20 is lysine, arginine, or glutamine;

Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

Xaa23 is isoleucine or valine;

Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, oraspartic acid; and

Xaa27 is leucine or lysine.

In still another specific embodiment, the peptide includes an amino acidsequence represented by General Formula 2 below:

Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General Formula 2, SEQ ID NO: 104)

In General Formula 2,

Xaa1 is 4-imidazoacetyl, histidine, or tyrosine;

Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

Xaa10 is tyrosine or cysteine;

Xaa13 is alanine, glutamine, tyrosine, or cysteine;

Xaa14 is leucine, methionine, or tyrosine;

Xaa15 is aspartic acid, glutamic acid, or leucine;

Xaa16 is glycine, glutamic acid, or serine;

Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, orlysine;

Xaa18 is alanine, glutamine, arginine, or histidine;

Xaa19 is alanine, glutamine, cysteine, or valine;

Xaa20 is lysine, glutamine, or arginine;

Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

Xaa23 is isoleucine or valine;

Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

Xaa28 is lysine, cysteine, asparagine, or aspartic acid;

Xaa29 is glycine, glutamine, cysteine, or histidine;

Xaa30 is cysteine, glycine, lysine, or histidine;

Xaa31 is proline or cysteine; and

Xaa40 is cysteine or is absent.

In still another specific embodiment, in General Formula 1,

Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

Xaa7 is threonine;

Xaa10 is tyrosine, cysteine, or valine;

Xaa12 is lysine or isoleucine;

Xaa13 is tyrosine, alanine, or cysteine;

Xaa14 is leucine or methionine;

Xaa15 is cysteine or aspartic acid;

Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;

Xaa18 is alanine, arginine, or histidine;

Xaa19 is alanine, glutamine, or cysteine;

Xaa20 is lysine or glutamine;

Xaa21 is glutamic acid, cysteine, or aspartic acid;

Xaa23 is valine;

Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and

Xaa27 is leucine or lysine.

In still another specific embodiment, in General Formula 2,

Xaa13 is alanine, tyrosine, or cysteine;

Xaa15 is aspartic acid or glutamic acid;

Xaa17 is glutamine, arginine, cysteine, or lysine;

Xaa18 is alanine, arginine, or histidine;

Xaa21 is cysteine, glutamic acid, glutamine, or aspartic acid;

Xaa23 is isoleucine or valine;

Xaa24 is cysteine, glutamine, or asparagine;

Xaa28 is cysteine, asparagine, or aspartic acid;

Xaa29 is glutamine, cysteine, or histidine; and

Xaa30 is cysteine, lysine, or histidine.

In still another specific embodiment, in General Formula 1,

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa7 is threonine;

Xaa10 is tyrosine or cysteine;

Xaa12 is lysine or isoleucine;

Xaa13 is tyrosine, alanine, or cysteine;

Xaa14 is leucine or methionine;

Xaa15 is cysteine or aspartic acid;

Xaa16 is glutamic acid;

Xaa17 is arginine, isoleucine, cysteine, or lysine;

Xaa18 is alanine, arginine, or histidine;

Xaa19 is alanine, glutamine, or cysteine;

Xaa20 is lysine or glutamine;

Xaa21 is glutamic acid or aspartic acid;

Xaa23 is valine;

Xaa24 is glutamine, asparagine, or aspartic acid;

Xaa27 is leucine; and

Xaa28 is cysteine, alanine, asparagine, or aspartic acid.

In still another specific embodiment, in General Formula 1,

Xaa1 is histidine or 4-imidazoacetyl;

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa3 is glutamine;

Xaa7 is threonine;

Xaa10 is tyrosine;

Xaa12 is isoleucine;

Xaa13 is alanine or cysteine;

Xaa14 is methionine;

Xaa15 is aspartic acid;

Xaa16 is glutamic acid;

Xaa17 is isoleucine or lysine;

Xaa18 is alanine or histidine;

Xaa19 is glutamine or cysteine;

Xaa20 is lysine;

Xaa21 is aspartic acid;

Xaa23 is valine;

Xaa24 is asparagine;

Xaa27 is leucine;

Xaa28 is alanine or asparagine;

Xaa29 is glutamine or threonine; and

Xaa30 is cysteine, or lysine, or is absent.

In still another specific embodiment,

in General Formula 1,

Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

Xaa3 is glutamine;

Xaa7 is threonine;

Xaa10 is tyrosine, cysteine, or valine;

Xaa12 is lysine;

Xaa13 is tyrosine;

Xaa14 is leucine;

Xaa15 is aspartic acid;

Xaa16 is glycine, glutamic acid, or serine;

Xaa17 is glutamine, arginine, cysteine, or lysine;

Xaa18 is alanine, arginine, or histidine;

Xaa19 is alanine or glutamine;

Xaa20 is lysine or glutamine;

Xaa21 is glutamic acid, cysteine, or aspartic acid;

Xaa23 is valine;

Xaa24 is alanine, glutamine, or cysteine;

Xaa27 is leucine or lysine; and

Xaa29 is glycine, glutamine, threonine, or histidine.

In still another specific embodiment, the peptide is a peptide includingan amino acid sequence represented by General Formula 3 below:

Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General Formula 3, SEQ ID NO: 105)

In General Formula 3,

Xaa1 is histidine or tyrosine;

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa13 is alanine, tyrosine, or cysteine;

Xaa17 is arginine, cysteine, or lysine;

Xaa18 is alanine or arginine;

Xaa19 is alanine or cysteine;

Xaa21 is glutamic acid or aspartic acid;

Xaa24 is glutamine or asparagine;

Xaa28 is cysteine or aspartic acid;

Xaa29 is cysteine, histidine, or glutamine;

Xaa30 is cysteine or histidine;

Xaa31 is proline or cysteine; and

Xaa40 is cysteine or is absent.

In still another specific embodiment, R1 is cysteine, CSSGQPPPS (SEQ IDNO: 109), GPSSGAPPPS (SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111),PSSGAPPPS (SEQ ID NO: 112), PSSGAPPPSG (SEQ ID NO: 113), PSSGAPPPSHG(SEQ ID NO: 114), PSSGAPPPSS (SEQ ID NO: 115), PSSGQPPPS (SEQ ID NO:116), or PSSGQPPPSC (SEQ ID NO: 117), or is absent.

In still another specific embodiment, in General Formulas 1 to 3, the16^(th) amino acid and the 20^(th) amino acid from the N-terminustogether form a ring.

In still another specific embodiment, the peptide includes an amino acidsequence selected from the group consisting of SEQ ID NOS: 1 to 102.

In still another specific embodiment, the peptide has at least one ofthe following activities of i) to iii):

i) activation of a GLP-1 receptor;

ii) activation of a glucagon receptor; and

iii) activation of a GIP receptor.

In still another specific embodiment, the peptide has an increased invivo half-life compared to that of any of native GLP-1, native glucagon,and native GIP.

In still another specific embodiment, the C-terminus of the peptide isamidated.

Another aspect of the present invention provides a polynucleotideencoding the isolated peptide, a recombinant expression vector includingthe polynucleotide, and a transformant including the polynucleotide orthe recombinant expression vector.

Still another aspect of the present invention provides a method forpreparing the isolated peptide.

Still another aspect of the present invention provides a compositionincluding the isolated peptide.

In a specific embodiment, the composition is a pharmaceuticalcomposition.

In another specific embodiment, the composition is for preventing ortreating metabolic syndrome.

In still another specific embodiment, the metabolic syndrome includesimpaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity,diabetes, hypertension, arteriosclerosis due to dyslipidemia,atherosclerosis, arteriosclerosis, or coronary heart disease.

Still another object of the present invention is to provide a method fortreating a target disease, which includes administering the isolatedpeptide or a composition containing the isolated peptide to a subject inneed thereof.

Still another object of the present invention is to provide a use of theisolated peptide or composition for use in the preparation of amedicament.

Advantageous Effects of the Invention

The peptide according to the present invention has activities to aglucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and aglucose-dependent insulinotropic polypeptide (GIP) receptor, and thuscan be applied for the treatment of metabolic syndrome.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detail.

Meanwhile, each of the explanations and exemplary embodiments disclosedherein can be applied to other explanations and exemplary embodiments.That is, all of the combinations of various factors disclosed hereinbelong to the scope of the present invention. Furthermore, the scope ofthe present invention should not be limited by the specific disclosureprovided hereinbelow.

Over the entire specification of the present invention, not only theconventional one-letter and three-letter codes for naturally occurringamino acids, but also those three-letter codes generally allowed forother amino acids, such as α-aminoisobutyric acid (Aib), Sar(N-methylglycine), and α-methyl-glutamic acid, are used.

Additionally, the amino acids mentioned herein are abbreviated accordingto the nomenclature rules of IUPAC-IUB as follows:

alanine (Ala, A) arginine (Arg, R) asparagine (Asn, N) aspartic acid(Asp, D) cysteine (Cys, C) glutamic acid (Glu, E) glutamine (Gln, Q)glycine (Gly, G) histidine (His, H) isoleucine (Ile, I) leucine (Leu, L)lysine (Lys, K) methionine (Met, M) phenylalanine (Phe, F) proline (Pro,P) serine (Ser, S) threonine (Thr, T) tryptophan (Trp, W) tyrosine (Tyr,Y) valine (Val, V)

An aspect of the present invention provides an isolated peptide whichhas activities to a glucagon receptor, a glucagon-like peptide-1 (GLP-1)receptor, and a glucose-dependent insulinotropic polypeptide (GIP)receptor.

In the present invention, the isolated peptide having activities to allof the glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor,and a glucose-dependent insulinotropic polypeptide (GIP) receptor can beused interchangeably with a triple agonist.

The triple agonist may include various materials (e.g., variouspeptides) which have a significant level of activities to glucagon,GLP-1, and GIP receptors.

The triple agonist having a significant level of activities to glucagon,GLP-1, and GIP receptors may exhibit in vitro activities of 0.1% orhigher, 1% or higher, 2% or higher, 3% or higher, 4% or higher, 5% orhigher, 6% or higher, 7% or higher, 8% or higher, 9% or higher, 10% orhigher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60%or higher, 70% or higher, 80% or higher, 90% or higher, and 100% orhigher, to one or more receptors, specifically two or more receptors,and more specifically all three of the receptors among the glucagon,GLP-1, and GIP receptors, compared to native ligands of thecorresponding receptors (native glucagon, native GLP-1, and native GIP),but is not particularly limited thereto.

The method for measuring the in vitro activities of the triple agonistmay refer to Example 2 of the present invention, but is not particularlylimited thereto.

Meanwhile, the triple agonist is characterized by having one or more ofthe following activities of i) to iii), specifically a significantactivity(-ies) thereof:

i) activation of a GLP-1 receptor; ii) activation of a glucagonreceptor; and iii) activation of a GIP receptor.

In particular, the activation of receptors may include, for example,those cases where the in vitro activities are 0.1% or higher, 1% orhigher, 2% or higher, 3% or higher, 4% or higher, 5% or higher, 6% orhigher, 7% or higher, 8% or higher, 9% or higher, 10% or higher, 20% orhigher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70%or higher, 80% or higher, 90% or higher, and 100% or higher, compared tonative ligands of the corresponding receptors, but the activation is notlimited thereto.

Additionally, the triple agonist may be one which has an increased invivo half-life relative to any one of native GLP-1, native glucagon, andnative GIP, but is not particularly limited thereto.

The above glucagon analog may be one which is non-naturally occurring,but is not particularly limited thereto.

Specifically, the isolated peptide may be an analog of native glucagon,but is not particularly limited thereto.

The native glucagon analog according to the present invention mayinclude peptides which have at least one difference in amino acidsequence compared to that of native glucagon; peptides which weremodified via modification of the native glucagon sequence; and mimeticsof the native glucagon.

Meanwhile, native glucagon may have the following amino acid sequence,but is not particularly limited thereto:

(SEQ ID NO: 118) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln- Trp-Leu-Met-Asn-Thr 

Specifically, the isolated peptide may be an analog of native glucagonwith a variation selected from the group consisting of substitution,addition, deletion, modification, and a combination thereof, on at leastone amino acid of the native glucagon sequence, but is not particularlylimited thereto.

Additionally, the substitution of the amino acid may include both asubstitution to an amino acid and a substitution to a non-nativecompound.

Additionally, the addition may be performed at the N-terminus and/orC-terminus of a peptide. Meanwhile, the length of the amino acid foraddition is not particularly limited, but 1 or more, 2 or more, 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more, and 11 or more amino acids may be added, and in a broadsense, the addition may include the addition of a polypeptide, but isnot particularly limited thereto.

More specifically, the glucagon analog may be those where 1 or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20amino acids selected from the group consisting of amino acids atpositions 1, 2, 3, 7, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23,24, 27, 28, and 29 in the amino acid sequence of native glucagon aresubstituted with other amino acids, and in addition, may be those where1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 ormore, 8 or more, 9 or more, 10 or more, or 11 or more amino acids areindependently or additionally added to the C-terminus thereof, but isnot particularly limited thereto.

Even more specifically, the glucagon analog may be those where 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more,8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 aminoacids selected from the group consisting of amino acids at positions 1,2, 3, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29in the amino acid sequence of native glucagon are substituted with otheramino acids, and in addition, may be those where 1 or more, 2 or more, 3or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 ormore, 10 or more, or 11 or more amino acids are independently oradditionally added to the C-terminus thereof, but is not particularlylimited thereto.

Even more specifically, the glucagon analog may be those where 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more,8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14or more, 15 or more, 16 or more, or 17 amino acids selected from thegroup consisting of amino acids at positions 1, 2, 3, 10, 13, 14, 15,16, 17, 18, 19, 20, 21, 23, 24, 28, and 29 in the amino acid sequence ofnative glucagon are substituted with other amino acids, and in addition,may be those where 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 ormore amino acids are independently or additionally added to theC-terminus thereof, but is not particularly limited thereto.

Even more specifically, the glucagon analog may be those where 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more,8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or14 amino acids selected from the group consisting of amino acids atpositions 1, 2, 13, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 inthe amino acid sequence of native glucagon are substituted with otheramino acids, and in addition, may be those where 1 or more, 2 or more, 3or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 ormore, 10 or more, or 11 or more amino acids are independently oradditionally added to the C-terminus thereof, but is not particularlylimited thereto.

The amino acids to be introduced into the above native glucagon may beselected from the group consisting of tyrosine, α-methyl-glutamic acid,Aib, methionine, glutamic acid, histidine, lysine, leucine, isoleucine,glutamine, valine, glycine, alanine, cysteine, serine, alanine, asparticacid, and arginine, but are not particularly limited thereto.

For example, the amino acid sequence(s) to be added may be at least oneamino acid sequence derived from a native GLP-1, native GIP, or nativeexendin-4 amino acid sequence.

The glucagon analog or triple agonist may include an intramolecularbridge (e.g., a covalent crosslinking or non-covalent crosslinking), andspecifically, is in a form including a ring, for example, is in a formwhere a ring is formed between the 16^(th) amino acid and the 20^(th)amino acid of the glucagon analog or the triple agonist, but is notparticularly limited thereto.

The non-limiting example of the ring may include a lactam bridge (or alactam ring).

Additionally, the glucagon analog or triple agonist includes all ofthose which are modified to include a ring, or include an amino acidcapable of forming a ring in a target position.

For example, the glucagon analog or triple agonist may be one where theamino acid pair of the 16^(th) and 20^(th) amino acids are substitutedwith glutamic acid or lysine, which can form a ring, respectively, butthe glucagon analog or triple agonist are not limited thereto.

The ring may be formed between amino acid side chains within theglucagon analog or triple agonist; for example, they may be in the formof a lactam ring between a side chain of lysine and a side chain ofglutamic acid, but the ring is not particularly limited thereto.

Examples of the glucagon analog prepared by a combination of thesemethods may include peptides, whose amino acid sequences differ fromthat of native glucagon in at least one amino acid, and in which theα-carbon in the N-terminus thereof is removed, while having activitiesto a glucagon receptor, a GLP-1 receptor, and a GIP receptor, etc., butare not limited thereto, and analogs of native glucagon applicable tothe present invention can be prepared by combining various methods forthe preparation of analogs.

Additionally, with respect to the triple agonist of the presentinvention, a part of the amino acids may be substituted with other aminoacids or non-natural compounds to avoid the recognition by peptidase forincreasing the in vivo half life of the triple agonist, but the tripleagonist is not particularly limited thereto.

Specifically, the peptide may be a peptide where the in vivo half lifewas increased by avoiding the recognition by the peptidase viasubstitution of the 2^(nd) amino acid sequence among the amino acidsequences of the triple agonist, but any substitution or modification ofamino acids to avoid the recognition by in vivo peptidase is includedwithout limitation.

Additionally, such modification for preparing analogs of native glucagonmay include all of the modifications using L-type or D-type amino acidsand/or non-natural amino acids; and/or a modification of nativesequence, for example, a modification of a side chain functional group,an intramolecular covalent bonding (e.g., a ring formation between sidechains), methylation, acylation, ubiquitination, phosphorylation,aminohexanation, biotinylation, etc.

Additionally, the modification may also include all of those where oneor more amino acids are added to the amino and/or carboxy terminus ofnative glucagon.

During the substitution or addition of amino acids, not only the 20amino acids commonly found in human proteins, but also atypical ornon-naturally occurring amino acids may be used. Commercial sources ofatypical amino acids may include Sigma-Aldrich, ChemPep Inc., andGenzyme Pharmaceuticals. The peptides including these amino acids andtypical peptide sequences may be synthesized and purchased fromcommercial suppliers, e.g., American Peptide Company, Bachem (USA), orAnygen (Korea).

Amino acid derivatives may be obtained in the same manner, and as onesuch example, 4-imidazoacetic acid may be used.

Additionally, the peptide according to the present invention may be inthe form of a variant where the amino and/or carboxy terminus, etc. ofthe peptide is chemically modified or protected by organic groups, oramino acids may be added to the terminus of the peptide, for itsprotection from proteases in vivo while increasing its stability.

In particular, in the case of a chemically-synthesized peptide, its N-and C-termini are electrically charged and thus the N- and C-termini ofthe peptide may be acetylated and/or amidated, but the peptide is notparticularly limited thereto.

Additionally, the peptide according to the present invention may includeall of those in the form of the peptide itself, a salt thereof (e.g., apharmaceutically acceptable salt thereof), or a solvate thereof.Additionally, the peptide may be in any pharmaceutically acceptableform.

The kind of the salt is not particularly limited. However, the salt ispreferably one that is safe and effective to a subject, e.g., a mammal,but is not particularly limited thereto.

The term “pharmaceutically acceptable” refers to a material which can beeffectively used for the intended use within the scope ofpharmaco-medical decision without inducing excessive toxicity,irritation, allergic responses, etc.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt derived from pharmaceutically acceptable inorganic acids, organicacids, or bases. Examples of the suitable salts may include hydrochloricacid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaricacid, maleic acid, phosphoric acid, glycolic acid, lactic acid,salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid,acetic acid, citric acid, methanesulfonic acid, formic acid, benzoicacid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid,etc. Examples of the salts derived from suitable bases may includealkali metals such as sodium, potassium, etc.; alkali earth metals suchas magnesium; ammonium, etc.

As used herein, the term “solvate” refers to a complex formed betweenthe peptide according to the present invention or a salt thereof and asolvent molecule.

In a specific embodiment, the peptide may be an isolated peptide whichincludes an amino acid sequence represented by General Formula 1 below.

Xaa-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General Formula 1, SEQ ID NO: 103)

In General Formula 1 above,

Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y);

Xaa2 is glycine (Gly, G), α-methyl-glutamic acid, or Aib(aminoisobutyric acid);

Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q);

Xaa7 is threonine (Thr, T) or isoleucine (Ile, I);

Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine(Cys, C), or valine (Val, V);

Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I);

Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), orcysteine (Cys, C);

Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y);

Xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic acid (Glu,E), or leucine (Leu, L);

Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S);

Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I),glutamic acid (Glu, E), cysteine (Cys, C), or lysine (Lys, K);

Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), orhistidine (His, H);

Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C), orvaline (Val, V);

Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);

Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L),cysteine (Cys, C), or aspartic acid (Asp, D);

Xaa23 is isoleucine (Ile, I) or valine (Val, V);

Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine (Cys, C),asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E);

Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K), ormethionine (Met, M);

Xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala, A),asparagine (Asn, N), or aspartic acid (Asp, D);

Xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine (Gln, Q),threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H);

Xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys, K), orhistidine (His, H), or is absent;

R1 is cysteine (Cys, C), GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent;

wherein,

m is -Cys-, -Pro-, or -Gly-Pro-;

n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent.

For example, the triple agonist may be one which includes an amino acidsequence selected from the group consisting of SEQ ID NOS: 1 to 102; andone which (essentially) consists of an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 1 to 102, but is not limitedthereto.

Additionally, although described as “a peptide consisting of aparticular SEQ ID NO” in the present invention, it does not exclude amutation that may occur by the addition of a meaningless sequenceupstream or downstream of the amino acid sequence of the correspondingSEQ ID NO, or a mutation that may occur naturally, or a silent mutationthereof, as long as the peptide has an activity the same as orcorresponding to that of the peptide which consists of an amino acidsequence of the corresponding SEQ ID NO, and even when the sequenceaddition or mutation is present, it obviously belongs to the scope ofthe present invention.

The above may be applicable in other specific embodiments or aspects ofthe present invention, but is not limited thereto.

Specifically, in General Formula above, Xaa14 may be leucine ormethionine, and Xaa15 may be cysteine, aspartic acid, or leucine.

Examples of the peptide may include a peptide which includes an aminoacid sequence selected from the group consisting of SEQ ID NOS: 1 to 12,14 to 17, and 21 to 102 or a peptide which (essentially) consists of thesame, but are not particularly limited thereto.

The peptide may significantly activate at least one of the glucagonreceptor, GLP-1 receptor, and GIP receptor, but is not particularlylimited thereto. Specifically, the peptide may be one whichsignificantly activates the GLP-1 receptor, or additionally the glucagonreceptor and/or GIP receptor, but is not particularly limited thereto.

Even more specifically, the peptide may be:

in General Formula 1 above,

Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

Xaa7 is threonine;

Xaa10 is tyrosine, cysteine, or valine;

Xaa12 is lysine or isoleucine;

Xaa13 is tyrosine, alanine, glutamine, or cysteine;

Xaa14 is leucine, cysteine, or methionine;

Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid;

Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, orlysine;

Xaa18 is alanine, glutamine, arginine, or histidine;

Xaa19 is alanine, glutamine, valine, or cysteine;

Xaa20 is lysine, arginine, or glutamine;

Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid;

Xaa23 is isoleucine or valine;

Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, oraspartic acid; and

Xaa27 is leucine or lysine, but is not particularly limited thereto.

Even more specifically,

in General Formula 1 above,

Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

Xaa7 is threonine;

Xaa10 is tyrosine, cysteine, or valine;

Xaa12 is lysine or isoleucine;

Xaa13 is tyrosine, alanine, or cysteine;

Xaa14 is leucine or methionine;

Xaa15 is cysteine or aspartic acid;

Xaa17 is glutamine, arginine, isoleucine, cysteine, or lysine;

Xaa18 is alanine, arginine, or histidine;

Xaa19 is alanine, glutamine, or cysteine;

Xaa20 is lysine or glutamine;

Xaa21 is glutamic acid, cysteine, or aspartic acid;

Xaa23 is valine;

Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; and

Xaa27 is leucine or lysine, but is not particularly limited thereto.

Even more specifically,

in General Formula 1 above,

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa7 is threonine;

Xaa10 is tyrosine or cysteine;

Xaa12 is lysine or isoleucine;

Xaa13 is tyrosine, alanine, or cysteine;

Xaa14 is leucine or methionine;

Xaa15 is cysteine or aspartic acid;

Xaa16 is glutamic acid;

Xaa17 is arginine, isoleucine, cysteine, or lysine;

Xaa18 is alanine, arginine, or histidine;

Xaa19 is alanine, glutamine, or cysteine;

Xaa20 is lysine or glutamine;

Xaa21 is glutamic acid or aspartic acid;

Xaa23 is valine;

Xaa24 is glutamine, asparagine, or aspartic acid;

Xaa27 is leucine; and

Xaa28 is cysteine, alanine, asparagine, or aspartic acid.

Specifically,

in General Formula 1 above,

Xaa1 is histidine or 4-imidazoacetyl;

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa3 is glutamine;

Xaa7 is threonine;

Xaa10 is tyrosine;

Xaa12 is isoleucine;

Xaa13 is alanine or cysteine;

Xaa14 is methionine;

Xaa15 is aspartic acid;

Xaa16 is glutamic acid;

Xaa17 is isoleucine or lysine;

Xaa18 is alanine or histidine;

Xaa19 is glutamine or cysteine;

Xaa20 is lysine;

Xaa21 is aspartic acid;

Xaa23 is valine;

Xaa24 is asparagine;

Xaa27 is leucine;

Xaa28 is alanine or asparagine;

Xaa29 is glutamine or threonine; and

Xaa30 is cysteine or lysine, or is absent.

More specifically,

in General Formula 1 above,

Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

Xaa3 is glutamine;

Xaa7 is threonine;

Xaa10 is tyrosine, cysteine, or valine;

Xaa12 is lysine;

Xaa13 is tyrosine;

Xaa14 is leucine;

Xaa15 is aspartic acid;

Xaa16 is glycine, glutamic acid, or serine;

Xaa17 is glutamine, arginine, cysteine, or lysine;

Xaa18 is alanine, arginine, or histidine;

Xaa19 is alanine or glutamine;

Xaa20 is lysine or glutamine;

Xaa21 is glutamic acid, cysteine, or aspartic acid;

Xaa23 is valine;

Xaa24 is alanine, glutamine, or cysteine;

Xaa27 is leucine or lysine; and

Xaa28 is glycine, glutamine, threonine, or histidine;

but is not particularly limited thereto.

These peptides may correspond to a case where the peptide hassignificant activation levels on both the GLP-1 receptor and glucagonreceptor, or higher activation levels compared to that on the GIPreceptor; a case where the peptide has significant activation levels onall of the GLP-1 receptor, glucagon receptor, and GIP receptor; or acase where the peptide has significant activation levels on both theGLP-1 receptor and GIP receptor and higher activation levels compared tothat on the glucagon receptor; but are not particularly limited thereto.

When the peptide has significant activation levels on both the GLP-1receptor and GIP receptor, and also higher activation levels compared tothat on the glucagon receptor, it is possible to provide a peptide withmore improved capability of controlling blood glucose levels along withthe effect of reducing body weight, whereas when the peptide hassignificant activation levels on all of the GLP-1 receptor, glucagonreceptor, and GIP receptor, there is an advantage in that the effect ofreducing body weight can be maximized, but the effects are notparticularly limited thereto.

Examples of the peptide may include a peptide which includes an aminoacid sequence selected from the group consisting of SEQ ID NOS: 8, 9, 21to 37, 39, 42, 43, 49 to 61, 64 to 83, 85, 86, 88, 89, 91 to 93, and 95to 102; or a peptide which (essentially) consists of the same, but arenot particularly limited thereto.

In a specific embodiment, the peptide may include an amino acid sequencerepresented by General Formula 2 below.

Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General Formula 2, SEQ ID NO: 104)

In General Formula 2 above,

Xaa1 is 4-imidazoacetyl, histidine, or tyrosine;

Xaa2 is glycine, α-methyl-glutamic acid, or Aib;

Xaa10 is tyrosine or cysteine;

Xaa13 is alanine, glutamine, tyrosine, or cysteine;

Xaa14 is leucine, methionine, or tyrosine;

Xaa15 is aspartic acid, glutamic acid, or leucine;

Xaa16 is glycine, glutamic acid, or serine;

Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, orlysine;

Xaa18 is alanine, glutamine, arginine, or histidine;

Xaa19 is alanine, glutamine, cysteine, or valine;

Xaa20 is lysine, glutamine, or arginine;

Xaa21 is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;

Xaa23 is isoleucine or valine;

Xaa24 is cysteine, alanine, glutamine, asparagine, or glutamic acid;

Xaa28 is lysine, cysteine, asparagine, or aspartic acid;

Xaa29 is glycine, glutamine, cysteine, or histidine;

Xaa30 is cysteine, glycine, lysine, or histidine;

Xaa31 is proline or cysteine; and

Xaa40 is cysteine or is absent.

More specifically, in General Formula 2 above,

Xaa13 is alanine, tyrosine, or cysteine;

Xaa15 is aspartic acid or glutamic acid;

Xaa17 is glutamine, arginine, cysteine, or lysine;

Xaa18 is alanine, arginine, or histidine;

Xaa21 is cysteine, glutamic acid, glutamine, or aspartic acid;

Xaa23 is isoleucine or valine;

Xaa24 is cysteine, glutamine, or asparagine;

Xaa28 is cysteine, asparagine, or aspartic acid;

Xaa29 is glutamine, cysteine, or histidine; and

Xaa30 is cysteine, lysine, or histidine.

Examples of the peptide may include a peptide which includes an aminoacid sequence selected from the group consisting of SEQ ID NOS: 21, 22,42, 43, 50, 64 to 77, and 95 to 102; more specifically, a peptide whichincludes an amino acid sequence selected from the group consisting ofSEQ ID NOS: 21, 22, 42, 43, 50, 64 to 77, and 96 to 102; or a peptidewhich (essentially) consists of the same, but are not particularlylimited thereto.

In a specific embodiment, the peptide may include an amino acid sequencerepresented by General Formula 3 below.

Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General Formula 3, SEQ ID NO: 105),

In General Formula 3 above,

Xaa1 is histidine or tyrosine;

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa13 is alanine, tyrosine or cysteine;

Xaa17 is arginine, cysteine, or lysine;

Xaa18 is alanine or arginine;

Xaa19 is alanine or cysteine;

Xaa21 is glutamic acid or aspartic acid;

Xaa24 is glutamine or asparagine,

Xaa28 is cysteine or aspartic acid;

Xaa29 is cysteine, histidine, or glutamine;

Xaa30 is cysteine or histidine;

Xaa31 is proline or cysteine; and

Xaa40 is cysteine or is absent.

Examples of the peptide may include a peptide which includes an aminoacid sequence selected from the group consisting of SEQ ID NOS: 21, 22,42, 43, 50, 64 to 71, 75 to 77, and 96 to 102; or a peptide which(essentially) consists of the same, but are not particularly limitedthereto.

Additionally, in General Formula 1 above, R1 may be cysteine,GKKNDWKHNIT (SEQ ID NO: 106), CSSGQPPPS (SEQ ID NO: 109), GPSSGAPPPS(SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111), PSSGAPPPS (SEQ ID NO:112), PSSGAPPPSG (SEQ ID NO: 113), PSSGAPPPSHG (SEQ ID NO: 114),PSSGAPPPSS (SEQ ID NO: 115), PSSGQPPPS (SEQ ID NO: 116), or PSSGQPPPSC(SEQ ID NO: 117), or is absent, but is not particularly limited thereto.

Still another aspect of the present invention provides a polynucleotideencoding the isolated peptide, a recombinant expression vector includingthe polynucleotide, and a transformant including the polynucleotide orthe recombinant expression vector.

The peptide is the same as explained above.

Additionally, the isolated polynucleotide encoding the peptide includes,within the scope of the present invention, a polynucleotide sequencehaving a sequence identity to the corresponding sequence of 75% orhigher, specifically 85% or higher, more specifically 90% or higher, andeven more specifically 95% or higher.

As used herein, the term “homology” indicates sequence similarity with awild-type amino acid sequence or wild-type nucleic acid sequence, andthe homology comparison may be done with the naked eye or using acommercially-available comparison program. Using a commerciallyavailable computer program, the homology between two or more sequencesmay be expressed as a percentage (%), and the homology (%) betweenadjacent sequences may be calculated.

As used herein, the term “recombinant vector” refers to a DNA constructin which the polynucleotide encoding the target peptide, e.g., thepeptide, is operably linked to an appropriate regulatory sequence toenable the expression of the target peptide, e.g., the peptide, in ahost cell.

The regulatory sequence includes a promoter capable of initiatingtranscription, any operator sequence for regulating the transcription, asequence encoding an appropriate mRNA ribosome-binding domain, and asequence for regulating the termination of transcription andtranslation. The recombinant vector, after being transformed into asuitable host cell, may be replicated or function irrespective of thehost genome, or may be integrated into the host genome itself.

The recombinant vector used in the present invention may not beparticularly limited as long as the vector is replicable in the hostcell, and it may be constructed using any vector known in the art.Examples of the vector conventionally used may include natural orrecombinant plasmids, cosmids, viruses, and bacteriophages. The vectorsto be used in the present invention are not particularly limited but anyexpression vector known in the art may be used.

The recombinant vector is used for the transformation of a host cell forproducing peptides of the present invention. Additionally, thesetransformed cells, as a part of the present invention, may be used foramplifying nucleic acid fragments and vectors, or they may be culturedcells or cell lines used in the recombinant production of peptides ofthe present invention.

As used herein, the term “transformation” refers to a process ofintroducing a recombinant vector including a polynucleotide encoding atarget protein into a host cell, thereby enabling the expression of theprotein encoded by the polynucleotide in the host cell. For thetransformed polynucleotide, it does not matter whether it is insertedinto the chromosome of a host cell and located thereon or locatedoutside of the chromosome, as long as it can be expressed in the hostcell, and both cases are included.

Additionally, the polynucleotide includes DNA and RNA which encode thetarget protein. The polynucleotide may be introduced in any form as longas it can be introduced into a host cell and expressed therein. Forexample, the polynucleotide may be introduced into a host cell in theform of an expression cassette, which is a gene construct including allof the essential elements required for self-expression. The expressioncassette may conventionally include a promoter operably linked to thepolynucleotide, a transcription termination signal, a ribosome-bindingdomain, and a translation termination signal. The expression cassettemay be in the form of an expression vector capable of self-replication.Additionally, the polynucleotide may be introduced into a host cell asit is and operably linked to a sequence essential for its expression inthe host cell, but is not limited thereto.

Additionally, as used herein, the term “operably linked” refers to afunctional connection between a promoter sequence, which initiates andmediates the transcription of the polynucleotide encoding the targetpeptide of the present invention, and the above gene sequence.

An appropriate host to be used in the present invention may not beparticularly limited as long as it can express the polynucleotide of thepresent invention. Examples of the appropriate host may include bacteriabelonging to the genus Escherichia such as E. coli; bacteria belongingto the genus Bacillus such as Bacillus subtilis; bacteria belonging tothe genus Pseudomonas such as Pseudomonas putida; yeasts such as Pichiapastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe;insect cells such as Spodoptera frugiperda (Sf9), and animal cells suchas CHO, COS, and BSC.

Still another aspect of the present invention provides a method forpreparing the isolated peptide.

The peptide is the same as explained above.

Additionally, the peptide of the present invention may be synthesized bya method well-known in the art, according to its length, e.g., by anautomatic peptide synthesizer, and may be produced by geneticengineering technology.

Specifically, the peptide of the present invention may be prepared by astandard synthesis method, a recombinant expression system, or any othermethod known in the art.

Accordingly, the peptide of the present invention may be synthesized bymany methods including, for example, the methods described below:

(a) a method of synthesizing a peptide by a solid-phase or liquid-phasemethod stepwise or by fragment assembly, followed by isolation andpurification of the final peptide product; or

(b) a method of expressing a nucleic acid construct encoding a peptidein a host cell and recovering the expression product from the host cellculture; or

(c) a method of performing an in vitro cell-free expression of a nucleicacid construct encoding a peptide and recovering the expression producttherefrom; or

a method of obtaining peptide fragments by any combination of themethods (a), (b), and (c), obtaining the peptide by linking the peptidefragments, and then recovering the peptide.

In a more specific example, a desired peptide may be produced by geneticmanipulation, which includes preparing a fusion gene encoding a fusionprotein, including a fusion partner and a peptide, transforming theresultant into a host cell, expressing the fusion protein, and cleavingthe peptide from the fusion protein using a protease or compoundfollowed by isolation. For this purpose, for example, a DNA sequenceencoding the amino acid sequence that can be cleaved by a protease suchas Factor Xa or enterokinase, CNBr, or a compound such as hydroxylamine,may be inserted between the fusion partner and a polynucleotide encodinga peptide.

Still another aspect of the present invention provides a compositioncontaining the isolated peptide.

The peptide is the same as explained above.

Specifically, the composition may be a pharmaceutical composition, andmore specifically, a pharmaceutical composition for preventing ortreating metabolic syndrome.

As used herein, the term “prevention” refers to all activities thatinhibit or delay metabolic syndrome by administering the above peptideor the composition, and the term “treatment” refers to all activitiesthat improve or advantageously change the symptoms of metabolic syndromeby administering the above peptide or the composition.

As used herein, the term “administration” refers to the introduction ofa particular substance into a subject by an appropriate method, and theadministration route of the composition may be any conventional routethat enables delivery of the composition to the target, for example,intraperitoneal administration, intravenous administration,intramuscular administration, subcutaneous administration, intradermaladministration, oral administration, topical administration, intranasaladministration, intrapulmonary administration, intrarectaladministration, etc., but is not limited thereto.

As used herein, the term “metabolic syndrome” refers to a symptom wherevarious diseases that occur due to chronic metabolic disorder occuralone or in combination. In particular, examples of diseases that belongto metabolic syndrome may include impaired glucose tolerance,hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension,arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis,and coronary heart disease, but are not limited thereto.

As used herein, the term “obesity” refers to a medical condition withexcess body fat accumulation and people are generally defined to beobese when their body mass index (BMI; a value of body mass (kg) overbody height squared (m)) is 25 or higher. Obesity is most commonlycaused by energy imbalance due to excessive food intake compared toenergy consumption over a long period of time. Obesity, being ametabolic disease that affects the entire body, increases thepossibility of developing of diabetes and hyperlipidemia, increases therisk of the incidence of sexual dysfunction, arthritis, andcardiovascular disease, and is associated with cancer development insome cases.

The pharmaceutical composition of the present invention may furthercontain a pharmaceutically acceptable carrier, excipient, or diluent.The pharmaceutically acceptable carrier, excipient, or diluent may benon-naturally occurring.

As used herein, the term “pharmaceutically acceptable” refers to theproperties of having a sufficient amount to exhibit a therapeutic effectand not causing adverse effects, and may be easily determined by askilled person in the art based on the factors well-known in the medicalfield, such as the kind of disease, age, body weight, health status,sex, drug sensitivity of a patient, administration route, administrationmethod, administration frequency, duration of treatment, a drug(s) to bemixed or administered simultaneously, etc.

The pharmaceutical composition of the present invention containing thepeptide of the present invention may further contain a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carrier may include,for oral administration, a binder, a lubricant, a disintegrant, anexcipient, a solubilizing agent, a dispersant, a stabilizing agent, asuspending agent, a coloring agent, a flavoring agent, etc.; forinjections, a buffering agent, a preserving agent, an analgesic, asolubilizing agent, an isotonic agent, a stabilizing agent, etc., whichmay be combined to be used; and for topical administrations, a base, anexcipient, a lubricant, a preserving agent, etc.

The formulation type of the composition according to the presentinvention may be prepared variously by combining with a pharmaceuticallyacceptable carrier described above. For example, for oraladministration, the composition may be formulated into tablets, troches,capsules, elixirs, suspensions, syrups, wafers, etc. For injections, thecomposition may be formulated into unit-dose ampoules or multi-dosecontainers. The composition may also be formulated into solutions,suspensions, tablets, pills, capsules, sustained-release formulations,etc.

Meanwhile, examples of suitable carriers, excipients, and diluents mayinclude lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calciumphosphate, calcium silicate, cellulose, methyl cellulose,microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate,mineral oil, etc. Additionally, the composition may further contain afiller, an anti-coagulant, a lubricant, a humectant, a flavoring agent,a preservative, etc.

Additionally, the pharmaceutical composition of the present inventionmay be prepared in any formulation type selected from the groupconsisting of tablets, pills, powders, granules, capsules, suspensions,liquid medicine for internal use, emulsions, syrups, sterile aqueoussolutions, non-aqueous solvents, lyophilized formulations, andsuppositories.

Additionally, the composition may be formulated into a unit dosage formsuitable for the patient's body, and is preferably formulated into apreparation useful for peptide drugs according to the typical method inthe pharmaceutical field so as to be administered by an oral orparenteral route, such as through skin, intravenously, intramuscularly,intraarterially, intramedullarily, intrathecally, intraventricularly,pulmonarily, transdermally, subcutaneously, intraperitoneally,intranasally, intragastrically, topically, sublingually, vaginally, orrectally, but is not limited thereto.

Additionally, the peptide may be used by mixing with variouspharmaceutically acceptable carriers such as physiological saline ororganic solvents. For increasing stability or absorptivity,carbohydrates such as glucose, sucrose, or dextrans, antioxidants suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins, or other stabilizers may be used as pharmaceutical drugs.

The administration dose and frequency of the pharmaceutical compositionof the present invention are determined by the type of activeingredient(s), together with various factors such as the disease to betreated, administration route, patient's age, gender, and body weight,and severity of the disease.

The total effective dose of the composition of the present invention maybe administered to a patient in a single dose or may be administered fora long period of time in multiple doses according to a fractionatedtreatment protocol. In the pharmaceutical composition of the presentinvention, the content of active ingredient(s) may vary depending on thedisease severity. Specifically, the total daily dose of the peptide ofthe present invention may be about 0.0001 mg to 500 mg per 1 kg of bodyweight of a patient. However, the effective dose of the peptide isdetermined considering various factors including patient's age, bodyweight, health conditions, gender, disease severity, diet, and excretionrate, in addition to administration route and treatment frequency of thepharmaceutical composition. In this regard, those skilled in the art mayeasily determine the effective dose suitable for the particular use ofthe pharmaceutical composition of the present invention. Thepharmaceutical composition according to the present invention is notparticularly limited to the formulation and administration route andmode, as long as it shows the effects of the present invention.

The pharmaceutical composition of the present invention shows excellentin vivo duration of efficacy and titer, and thus the number andfrequency of administration of the pharmaceutical preparation of thepresent invention can be significantly reduced.

Still another aspect of the present invention provides a method fortreating a target disease, which includes administering the isolatedpeptide or a composition containing the isolated peptide to a subject inneed thereof. The target disease may be a metabolic syndrome.

The isolated peptide, or the composition containing the same, themetabolic syndrome and treatment thereof are the same as explainedabove.

As used herein, the term “subject” refers to a subject suspected ofhaving a metabolic syndrome, and the subject suspected of having ametabolic syndrome refers to mammals including humans, rats, cattle,etc., which have or are at the risk of developing the metabolicsyndrome, but any subject which can be treated with the above peptide ofthe present invention or the composition containing the same is includedwithout limitation.

The therapeutic method of the present invention may includeadministering a pharmaceutically effective amount of the pharmaceuticalcomposition containing the peptide. The total daily dose of thecomposition may be determined within the scope of appropriate medicaljudgment by a physician, and the composition may be administered once orseveral times in divided doses a day. However, for the purpose of thepresent invention, the specific therapeutically effective dose of thecomposition for any particular patient is preferably applied differentlydepending on various factors including the kind and degree of responseto be achieved, specific compositions including whether other agents areoccasionally used therewith, the patient's age, body weight, healthcondition, gender and diet, the time and route of administration, thesecretion rate of the composition, the duration of treatment, otherdrugs used in combination or simultaneously with the specificcompositions, and similar factors well-known in the medical field.

Still another aspect of the present invention provides the use of theisolated peptide or composition containing the peptide in thepreparation of a medicament. The medicament may be for preventing ortreating metabolic syndrome.

The isolated peptide or composition and metabolic syndrome are the sameas explained above.

Hereinafter, the present invention will be described in more detail withreference to the following Examples. However, these Examples are forillustrative purposes only and the invention is not intended to belimited by these Examples.

Example 1: Preparation of Triple Agonists

Triple agonists showing activities to all of GLP-1, GIP, and glucagonreceptors were prepared and their amino acid sequences are shown inTable 1 below.

TABLE 1 SEQ ID NO Sequence Information 1H X Q G T F T S D V S S Y L D G Q A A K E F I A W L V K G C  2H X Q G T F T S D V S S Y L D G Q A Q K E F I A W L V K G C  3H X Q G T F T S D V S S Y L L G Q A A K Q F I A W L V K G G G P S S G A P P P S C  4H X Q G T F T S D V S S Y L L G Q Q Q K E F I A W L V K G C  5H X Q G T F T S D V S S Y L L G Q Q Q K E F I A W L V K G G G P S S G A P P P S C  6H X Q G T F T S D V S S Y L D G Q A A K E F V A W L L K G C  7H X Q G T F T S D V S K Y L D G Q A A K E F V A W L L K G C  8H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L K G C  9H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L A G C  10H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L A G G G P S S G A P P P S C  11C A G E G T F T S D L S K Y L D S R R Q Q L F V Q W L K A G G P S S G A P P P S H G  12C A G E G T F I S D L S K Y M D E Q A V Q L F V E W L M A G G P S S G A P P P S H G  13C A G E G T F I S D Y S I Q L D E I A V Q D F V E W L L A Q K P S S G A P P P S H G  14C A G Q G T F T S D Y S I Q L D E I A V R D F V E W L K N G G P S S G A P P P S H G  15C A G Q G T F T S D L S K Q M D E E A V R L F I E W L K N G G P S S G A P P P S H G  16C A G Q G T F T S D L S K Q M D S E A Q Q L F I E W L K N G G P S S G A P P P S H G  17C A G Q G T F T S D L S K Q M D E E R A R E F I E W L L A Q K P S S G A P P P S H G  18C A G Q G T F T S D L S K Q M D S E R A R E F I E W L K N T G P S S G A P P P S H G  19C A G Q G T F T S D L S I Q Y D S E H Q R D F I E W L K D T G P S S G A P P P S H G  20C A G Q G T F T S D L S I Q Y E E E A Q Q D F V E W L K D T G P S S G A P P P S H G  21Y X Q G T F T S D Y S K Y L D E C R A K E F V Q W L L D H H P  RingS S G Q P P P S  formation 22Y X Q G T F T S D Y S K C L D E K R A K E F V Q W L L D H H P  RingS S G Q P P P S  formation 23Y X Q G T F T S D Y S K Y L D E C R A K E F V Q W L L A Q K G  RingK K N D W K H N I T  formation 24Y X Q G T F T S D Y S K Y L D E C R A K E F V Q W L K N G G P  RingS S G A P P P S  formation 25H X Q G T F T S D C S K Y L D E R A A Q D F V Q W L L D G G P S S G A P P P S  26H X Q G T F T S D C S K Y L D S R A A Q D F V Q W L L D G G P S S G A P P P S  27H X Q G T F T S D Y S K Y L D E R A C Q D F V Q W L L D Q G G P S S G A P P P S  28H X Q G T F T S D Y S K Y L D E K R A Q E F V C W L L A Q K G K K N D W K H N I T  29H X Q G T F T S D Y S K Y L D E K A A K E F V Q W L L N T C  Ringformation 30H X Q G T F T S D Y S K Y L D E K A Q K E F V Q W L L D T C  Ringformation 31 H X Q G T F T S D Y S K Y L D E K A C K E F V Q W L L A Q Ring formation 32H X Q G T F T S D Y S K Y L D E K A C K D F V Q W L L D G G P  RingS S G A P P P S  formation 33H X Q G T F T S D Y S I A M D E I H Q K D F V N W L L A Q K C  Ringformation 34H X Q G T F T S D Y S K Y L D E K R Q K E F V N W L L A Q K C  Ringformation 35H X Q G T F T S D Y S I A M D E I H Q K D F V N W L L N T K C  Ringformation 36H X Q G T F T S D Y S K Y L C E K R Q K E F V Q W L L N G G P  RingS S G A P P P S G  formation 37H X Q G T F T S D Y S K Y L D E C R Q K E F V Q W L L N G G P  RingS S G A P P P S G  formation 38C A X Q G T F T S D K S S Y L D E R A A Q D F V Q W L L D G G P S S G A P P P S S  39H X Q G T F T S D Y S K Y L D G Q H A Q C F V A W L L A G G G P S S G A P P P S  40H X Q G T F T S D K S K Y L D E R A C Q D F V Q W L L D G G P S S G A P P P S  41H X Q G T F T S D K S K Y L D E C A A Q D F V Q W L L D G G P S S G A P P P S  42Y X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L D H H P  RingS S G Q P P P S C  formation 43Y X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L D H H C  RingS S G Q P P P S  formation 44H G Q G T F T S D C S K Q L D G Q A A Q E F V A W L L A G G P S S G A P P P S  45H G Q G T F T S D C S K Y M D G Q A A Q D F V A W L L A G G P S S G A P P P S  46H G Q G T F T S D C S K Y L D E Q H A Q E F V A W L L A G G P S S G A P P P S  47H G Q G T F T S D C S K Y L D G Q R A Q E F V A W L L A G G P S S G A P P P S  48H G Q G T F T S D C S K Y L D G Q R A Q D F V N W L L A G G P S S G A P P P S  49C A X Q G T F T S D Y S I C M D E I H Q K D F V N W L L N T K  Ringformation 50H X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L D H H P  RingS S G Q P P P S C  formation 51H X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L N T C  Ringformation 52H X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L D T C  Ringformation 53H X E G T F T S D Y S I A M D E I H Q K D F V N W L L A Q C  Ringformation 54H X E G T F T S D Y S I A M D E I H Q K D F V D W L L A E C  Ringformation 55H X Q G T F T S D Y S I A M D E I H Q K D F V N W L L A Q C  Ringformation 56H X Q G T F T S D Y S K Y L D E K R Q K E F V N W L L A Q C  Ringformation 57H X Q G T F T S D Y S I A M D E I H Q K D F V N W L L N T C  Ringformation 58H X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L N T K C  Ringformation 59C A X Q G T F T S D Y S I C M D E K H Q K D F V N W L L N T K  Ringformation 60C A X Q G T F T S D Y S I A M D E K H C K D F V N W L L N T K  Ringformation 61C A X Q G T F T S D Y S I A M D E I A C K D F V N W L L N T K  Ringformation 62C A X Q G T F T S D K S K Y L D E R A A Q D F V Q W L L D G G P S S G A P P P S  63C A X Q G T F T S D C S K Y L D E R A A Q D F V Q W L L D G G P S S G A P P P S  64Y X Q G T F T S D Y S K Y L D E C A A K E F V Q W L L D H H P  RingS S G Q P P P S  formation 65H X Q G T F T S D Y S K C L D E K R A K E F V Q W L L D H H P  RingS S G Q P P P S  formation 66Y X Q G T F T S D Y S K Y L D E C R A K D F V Q W L L D H H P  RingS S G Q P P P S  formation 67Y X Q G T F T S D Y S K Y L D E C A A K D F V Q W L L D H H P  RingS S G Q P P P S  formation 68Y X Q G T F T S D Y S K C L D E K A A K E F V Q W L L D H H P  RingS S G Q P P P S  formation 69Y X Q G T F T S D Y S K C L D E R A A K E F V Q W L L D H H P  RingS S G Q P P P S  formation 70Y X Q G T F T S D Y S K C L D E K R A K D F V Q W L L D H H P  RingS S G Q P P P S  formation 71Y X Q G T F T S D Y S K Y L D E R A C K D F V Q W L L D H H P  RingS S G Q P P P S  formation 72Y X Q G T F T S D C S K Y L D E R A A K D F V Q W L L D H H P  RingS S G Q P P P S  formation 73C A X Q G T F T S D Y S K Y L D E C R A K E F V Q W L L D H H  RingP S S G Q P P P S  formation 74C A X Q G T F T S D Y S K C L D E K R A K E F V Q W L L D H H  RingP S S G Q P P P S  formation 75Y X Q G T F T S D Y S K Y L D E K A A K E F V Q W L L D H H P  RingS S G Q P P P S C  formation 76Y X Q G T F T S D Y S K Y L D E K R A K D F V Q W L L D H H P  RingS S G Q P P P S C  formation 77Y X Q G T F T S D Y S K Y L D E K A A K D F V Q W L L D H H P  RingS S G Q P P P S C  formation 78H X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L D T K C  Ringformation 79H X E G T F T S D Y S I A M D E I H Q K D F V N W L L A Q K C  Ringformation 80H X E G T F T S D Y S I A M D E I H Q K D F V D W L L A E K C  Ringformation 81C A X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L N T C  Ringformation 82C A X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L D T C  Ringformation 83C A X E G T F T S D Y S I A M D E I H Q K D F V N W L L A Q C  Ringformation 84C A X E G T F T S D Y S I A M D E I H Q K D F V D W L L A E C  Ringformation 85C A X Q G T F T S D Y S I A M D E I H Q K D F V N W L L A Q C  Ringformation 86C A X Q G T F T S D Y S K Y L D E K R Q K E F V N W L L A Q C  Ringformation 87C A X Q G T F T S D Y S I A M D E I H Q K D F V N W L L N T C  Ringformation 88C A X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L N T K  Ring C formation 89C A X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L D T K  Ring C formation 90C A X E G T F T S D Y S I A M D E I H Q K D F V N W L L A Q K  Ring C formation 91C A X E G T F T S D Y S I A M D E I H Q K D F V D W L L A E K C  Ringformation 92C A X Q G T F T S D Y S I A M D E I H Q K D F V N W L L A Q K  Ring C formation 93C A X Q G T F T S D Y S K Y L D E K R Q K E F V N W L L A Q K  Ring C formation 94C A X Q G T F T S D Y S I A M D E I H Q K D F V N W L L N T K  Ring C formation 95Y X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L C H H P  RingS S G Q P P P S  formation 96Y X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L D H C P  RingS S G Q P P P S  formation 97Y X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L D C H P  RingS S G Q P P P S  formation 98Y X Q G T F T S D Y S K A L D E K A A K E F V N W L L D H H P  RingS S G Q P P P S C  formation 99Y X Q G T F T S D Y S K A L D E K A A K D F V N W L L D H H P  RingS S G Q P P P S C  formation 100Y X Q G T F T S D Y S K A L D E K A A K E F V Q W L L D Q H P  RingS S G Q P P P S C  formation 101Y X Q G T F T S D Y S K A L D E K A A K E F V N W L L D Q H P  RingS S G Q P P P S C  formation 102Y X Q G T F T S D Y S K A L D E K A A K D F V N W L L D Q H P  RingS S G Q P P P S C  formation

In the sequences described in Table 1, the amino acid represented by Xrepresents aminoisobutyric acid (Aib), which is a non-natural aminoacid, and the underlined amino acids represent the formation of a ringbetween the underlined amino acids. Additionally, in Table 1, CArepresents 4-imidazoacetyl and Y represents tyrosine.

Example 2: Measurement of In Vitro Activities of Triple Agonists

The activities of the triple agonists prepared in Example 1 weremeasured by a method of measuring in vitro cellular activities usingcell lines, where a GLP-1 receptor, a glucagon (GCG) receptor, and a GIPreceptor are transformed, respectively.

Each of the cell lines above is one in which the genes for human GLP-1receptor, human GCG receptor, and human GIP receptor were transformed inChinese hamster ovary (CHO), respectively, and can be expressed therein,and is thus suitable for the measurement of the activities of GLP-1,GCG, and GIP. Accordingly, the activity for each part was measured usingthe respective transformed cell line.

For the measurement of the GLP-1 activity of the triple agonistsprepared in Example 1, human GLP-1 was subjected to a 4-fold serialdilution from 50 nM to 0.000048 nM and the triple agonists prepared inExample 1 were subjected to a 4-fold serial dilution from 400 nM to0.00038 nM.

The culture solution was removed from the cultured CHO cells, in whichthe human GLP-1 receptor was expressed, and each of the serially-dilutedmaterials was added to the CHO cells in an amount of 5 μL, respectively.Then, a buffer solution containing cAMP antibody was added thereto in anamount of 5 μL and cultured at room temperature for 15 minutes. Then, adetection mix containing a cell lysis buffer was added thereto in anamount of 10 μL for the lysis of the cells and reacted at roomtemperature for 90 minutes. The cell lysates, upon completion of thereaction, were applied to LANCE cAMP kit (PerkinElmer, USA) to calculatethe EC₅₀ value via accumulated cAMP, and the values were compared witheach other. The relative potencies compared to human GLP-1 are shown inTable 2 below.

For the measurement of the GCG activity of the triple agonists preparedin Example 1, human GCG was subjected to a 4-fold serial dilution from50 nM to 0.000048 nM and the triple agonists prepared in Example 1 weresubjected to a 4-fold serial dilution from 400 nM to 0.00038 nM.

The culture solution was removed from the cultured CHO cells, in whichthe human GCG receptor was expressed, and each of the serially-dilutedmaterials was added to the CHO cells in an amount of 5 μL, respectively.Then, a buffer solution containing cAMP antibody was added thereto in anamount of 5 μL and cultured at room temperature for 15 minutes. Then, adetection mix containing a cell lysis buffer was added thereto in anamount of 10 μL for the lysis of the cells and reacted at roomtemperature for 90 minutes. The cell lysates, upon completion of thereaction, were applied to LANCE cAMP kit (PerkinElmer, USA) to calculatethe EC₅₀ value via accumulated cAMP, and the values were compared witheach other. The relative potencies compared to human GCG are shown inTable 2 below.

For the measurement of the GIP activity of the triple agonists preparedin Example 1, human GIP was subjected to a 4-fold serial dilution from50 nM to 0.000048 nM and the triple agonists prepared in Example 1 weresubjected to a 4-fold serial dilution from 400 nM to 0.00038 nM.

The culture solution was removed from the cultured CHO cells, in whichthe human GIP receptor was expressed, and each of the serially-dilutedmaterials was added to the CHO cells in an amount of 5 μL, respectively.Then, a buffer solution containing cAMP antibody was added thereto in anamount of 5 μL and cultured at room temperature for 15 minutes. Then, adetection mix containing a cell lysis buffer was added thereto in anamount of 10 μL for the lysis of the cells and reacted at roomtemperature for 90 minutes. The cell lysates, upon completion of thereaction, were applied to LANCE cAMP kit (PerkinElmer, USA) to calculatethe EC₅₀ value via accumulated cAMP, and the values were compared witheach other. The relative potencies compared to human GIP are shown inTable 2 below.

TABLE 2 In vitro activity compared to native peptide (%) SEQ ID NO vs.GLP-1 vs. Glucagon vs. GIP 1 3.2 <0.1 <0.1 2 5.9 <0.1 <0.1 3 1.8 <0.1<0.1 4 8.5 <0.1 <0.1 5 42.1 <0.1 <0.1 6 17.0 <0.1 <0.1 7 13.7 <0.1 <0.18 14.2 0.10 <0.1 9 32.1 0.13 <0.1 10 46.0 <0.1 <0.1 11 1.4 <0.1 <0.1 120.4 <0.1 <0.1 13 <0.1 <0.1 <0.1 14 28.0 <0.1 <0.1 15 79.2 <0.1 <0.1 162.1 <0.1 <0.1 17 0.2 <0.1 <0.1 18 <0.1 <0.1 <0.1 19 <0.1 <0.1 <0.1 20<0.1 <0.1 <0.1 21 17.8 267 22.7 22 20.1 140 59.7 23 4.01 9.3 <0.1 2441.2 9.3 <0.1 25 82.6 0.1 <0.1 26 64.5 0.2 <0.1 27 83.1 0.8 0.9 28 17.21.6 <0.1 29 38.5 6.0 <0.1 30 142 0.7 0.8 31 135 2.2 2.4 32 151 1.7 8.833 24.5 <0.1 10.4 34 19.1 0.92 0.6 35 7.5 <0.1 1.3 36 37.4 0.39 0.2 37236 6.21 2.2 38 2.3 — — 39 13.9 0.53 <0.1 40 75.2 <0.1 <0.1 41 34.3 <0.1<0.1 42 33.9 205.8 7.8 43 12.6 88.4 3.70 44 1.3 <0.1 <0.1 45 6.6 <0.1<0.1 46 1.4 <0.1 <0.1 47 2.4 <0.1 <0.1 48 1.5 <0.1 <0.1 49 29.8 <0.1 3.350 67.4 50.5 2.7 51 14.4 2.0 0.1 52 44.1 7.5 0.3 53 161 8.4 1.3 54 30.61.4 0.1 55 27.1 0.7 2.4 56 57.9 4.9 0.8 57 11.7 <0.1 0.3 58 39.1 2.6 0.259 40.3 <0.1 4.0 60 106.2 <0.1 8.2 61 59.8 <0.1 2.8 62 5.2 <0.1 <0.1 6315.3 <0.1 <0.1 64 64.6 60.1 92.9 65 95.4 25.2 11.6 66 15.8 172 17.2 6728.5 46.2 39.8 68 27.9 8.8 107 69 24.3 9.6 62.8 70 15.1 71.3 64.4 7190.1 12.7 94.7 72 11.5 1.0 1.6 73 22.6 5.4 3.0 74 12.9 0.9 1.0 75 35.18.5 18.0 76 10.3 47.6 11.7 77 38.7 12.2 35.5 78 51.0 14.0 0.12 79 41.54.9 1.4 80 8.1 0.0 0.1 81 7.8 0.3 <0.1 82 9.5 1.1 <0.1 83 47.3 1.3 0.484 4.2 <0.1 <0.1 85 4.3 <0.1 0.3 86 28.4 0.4 0.2 87 0.9 <0.1 <0.1 88 9.60.3 <0.1 89 7.1 0.7 <0.1 90 7.4 <0.1 <0.1 91 31.9 16.8 0.3 92 0.8 <0.10.4 93 5.7 0.3 0.7 94 0.5 <0.1 <0.1 95 2.1 0.4 <0.1 96 34.4 194.8 5.2 9710.5 62.8 2.6 98 28.1 8.2 47.1 99 20.9 14.9 57.7 100 42.2 12.7 118.5 10123.2 13.9 40.1 102 23.3 29.5 58.0

The novel glucagon analogs prepared above have the function of tripleagonists which can activate all of GLP-1 receptors, GIP receptors, andglucagon receptors, and thus the glucagon analogs can be used as atherapeutic material for treating patients with metabolic syndromeincluding diabetes and obesity.

From the foregoing, a skilled person in the art to which the presentinvention pertains will be able to understand that the present inventionmay be embodied in other specific forms without modifying the technicalconcepts or essential characteristics of the present invention. In thisregard, the exemplary embodiments disclosed herein are only forillustrative purposes and should not be construed as limiting the scopeof the present invention. On the contrary, the present invention isintended to cover not only the exemplary embodiments but also variousalternatives, modifications, equivalents, and other embodiments that maybe included within the spirit and scope of the present invention asdefined by the appended claims.

1-21. (canceled)
 22. An isolated peptide having activities to a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insulinotropic polypeptide (GIP) receptor, wherein the peptide comprises an amino acid sequence of the following Formula (1): (SEQ ID NO: 105) Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys- Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe- Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser- Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40

wherein, in Formula (1), Xaa1 is His or Tyr; Xaa2 is α-methyl-glutamic acid or aminoisobutyric acid (Aib); Xaa13 is Ala, Tyr, or Cys; Xaa17 is Arg, Cys, or Lys; Xaa18 is Ala or Arg; Xaa19 is Ala or Cys; Xaa21 is Glu or Asp; Xaa24 is Gln or Asn; Xaa28 is Cys or Asp; Xaa29 is Cys, His, or Gln; Xaa30 is Cys or His; Xaa31 is Pro or Cys; and Xaa40 is Cys or is absent.
 23. The isolated peptide of claim 22, which is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97 and
 100. 24. The isolated peptide according to claim 22, wherein, in Formula 1, the 16^(th) amino acid and the 20^(th) amino acid from the N-terminus together form a ring.
 25. The isolated peptide of claim 22, wherein Xaa1 is Tyr.
 26. An isolated peptide having activities to a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insulinotropic polypeptide (GIP) receptor, wherein the peptide comprises an amino acid sequence of the following Formula (1): (SEQ ID NO: 103) Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser- Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19- Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28- Xaa29-Xaa30-R1

wherein, in Formula (1), Xaa1 is His, 4-imidazoacetyl (CA), or Tyr; Xaa2 is α-methyl-glutamic acid, or aminoisobutyric acid (Aib); Xaa3 is Gln; Xaa7 is Thr; Xaa10 is Tyr; Xaa12 is Lys; Xaa13 is Tyr, Ala, or Cys; Xaa14 is Leu or Met; Xaa15 is Cys, Asp, or Glu; Xaa16 is Gly or Glu; Xaa17 is Gln, Arg, Be, Glu, Cys, or Lys; Xaa18 is Ala, Arg, or His; Xaa19 is Ala, Gln, or Cys; Xaa20 is Lys or Gln; Xaa21 is Glu, Gln, Cys, or Asp; Xaa23 is Ile or Val; Xaa24 is Ala, Gln, Cys, or Asn; Xaa27 is Leu or Lys; Xaa28 is Cys, Lys, Ala, Asn, or Asp; Xaa29 is Cys, Gly, Gln, Thr, Glu, or His; Xaa30 is Cys, Gly, Lys, or His, or is absent; and R1 is Cys, GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent; wherein m is -Cys-, -Pro-, or -Gly-Pro-; and n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent, and wherein the peptide has an increased in vitro half-life compared to that of native GLP-1, native glucagon, and native GIP.
 27. The isolated peptide of claim 26, wherein the peptide exhibits at each of glucagon receptor, GLP-1 receptor and GIP receptor an in vitro activity of 1% or higher compared to that of the respective native ligand.
 28. The isolated peptide of claim 26, wherein R1 is GKKNDWKHNIT (SEQ ID NO: 106).
 29. The isolated peptide of claim 26, wherein X is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 21 to 24, 27 to 32, 34, 36, 37, 39, 42, 43, 50 to 52, 56, 58, 64 to 71, 73 to 78, 81, 82, 86, 88, 89, 93, and 95 to
 102. 30. The isolated peptide of claim 26, wherein X is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 21, 22, 23, 31, 32, 42, 43, 50, 53, 55, 64 to 77, 79, and 96 to
 102. 31. The isolated peptide of claim 26, wherein, in Formula (1), Xaa13 is Ala, Tyr, or Cys; Xaa15 is Asp or Glu; Xaa17 is Gln, Arg, Cys, or Lys; Xaa18 is Ala, Arg, or His; Xaa21 is Cys, Glu, Gln, or Asp; Xaa23 is Ile or Val; Xaa24 is Cys, Gln, or Asn; Xaa28 is Cys, Asn, or Asp; Xaa29 is Gln, Cys, or His; and Xaa30 is Cys, Lys, or His.
 32. The isolated peptide of claim 26, wherein, in Formula (1), Xaa1 is His or 4-imidazoacetyl; Xaa13 is Ala or Cys; Xaa14 is Met; Xaa15 is Asp; Xaa16 is Glu; Xaa17 is Ile or Lys; Xaa18 is Ala or His; Xaa19 is Gln or Cys; Xaa20 is Lys; Xaa21 is Asp; Xaa23 is Val; Xaa24 is Asn; Xaa28 is Ala or Asn; Xaa29 is Gln or Thr; and Xaa30 is Cys or Lys, or is absent.
 33. A pharmaceutical composition comprising the peptide of claim
 22. 34. A method of treating metabolic syndrome comprising administering the peptide according to claim 22 to a subject in need thereof.
 35. The method of claim 34, wherein the metabolic syndrome comprises impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, or coronary heart disease.
 36. A pharmaceutical composition comprising the peptide of claim
 26. 37. A method of treating metabolic syndrome comprising administering the peptide according to claim 26 to a subject in need thereof.
 38. The method of claim 37, wherein the metabolic syndrome comprises impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, or coronary heart disease. 